Targeting the innate immune system in pediatric and adult AML

While the introduction of T cell-based immunotherapies has improved outcomes in many cancer types, the development of immunotherapies for both adult and pediatric AML has been relatively slow and limited. In addition to the need to identify suitable target antigens, a better understanding of the immunosuppressive tumor microenvironment is necessary for the design of novel immunotherapy approaches. To date, most immune characterization studies in AML have focused on T cells, while innate immune lineages such as monocytes, granulocytes and natural killer (NK) cells, received less attention. In solid cancers, studies have shown that innate immune cells, such as macrophages, myeloid-derived suppressor cells and neutrophils are highly plastic and may differentiate into immunosuppressive cells depending on signals received in their microenvironment, while NK cells appear to be functionally impaired. Hence, an in-depth characterization of the innate immune compartment in the TME is urgently needed to guide the development of immunotherapeutic interventions for AML. In this review, we summarize the current knowledge on the innate immune compartment in AML, and we discuss how targeting its components may enhance T cell-based- and other immunotherapeutic approaches.</p

Targeting the innate immune system in pediatric and adult AML

While the introduction of T cell-based immunotherapies has improved outcomes in many cancer types, the development of immunotherapies for both adult and pediatric AML has been relatively slow and limited. In addition to the need to identify suitable target antigens, a better understanding of the immunosuppressive tumor microenvironment is necessary for the design of novel immunotherapy approaches. To date, most immune characterization studies in AML have focused on T cells, while innate immune lineages such as monocytes, granulocytes and natural killer (NK) cells, received less attention. In solid cancers, studies have shown that innate immune cells, such as macrophages, myeloid-derived suppressor cells and neutrophils are highly plastic and may differentiate into immunosuppressive cells depending on signals received in their microenvironment, while NK cells appear to be functionally impaired. Hence, an in-depth characterization of the innate immune compartment in the TME is urgently needed to guide the development of immunotherapeutic interventions for AML. In this review, we summarize the current knowledge on the innate immune compartment in AML, and we discuss how targeting its components may enhance T cell-based- and other immunotherapeutic approaches.</p

Serum response factor is crucial for actin cytoskeletal organization and focal adhesion assembly in embryonic stem cells

The activity of serum response factor (SRF), an essential transcription factor in mouse gastrulation, is regulated by changes in actin dynamics. Using Srf(−/−) embryonic stem (ES) cells, we demonstrate that SRF deficiency causes impairments in ES cell spreading, adhesion, and migration. These defects correlate with defective formation of cytoskeletal structures, namely actin stress fibers and focal adhesion (FA) plaques. The FA proteins FA kinase (FAK), β1-integrin, talin, zyxin, and vinculin were downregulated and/or mislocalized in ES cells lacking SRF, leading to inefficient activation of the FA signaling kinase FAK. Reduced overall actin expression levels in Srf(−/−) ES cells were accompanied by an offset treadmilling equilibrium, resulting in lowered F-actin levels. Expression of active RhoA-V14 rescued F-actin synthesis but not stress fiber formation. Introduction of constitutively active SRF-VP16 into Srf(−/−) ES cells, on the other hand, strongly induced expression of FA components and F-actin synthesis, leading to a dramatic reorganization of actin filaments into stress fibers and lamellipodia. Thus, using ES cell genetics, we demonstrate for the first time the importance of SRF for the formation of actin-directed cytoskeletal structures that determine cell spreading, adhesion, and migration. Our findings suggest an involvement of SRF in cell migratory processes in multicellular organisms

NUP98 oncofusions in myeloid malignancies:An update on molecular mechanisms and therapeutic opportunities

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a heterogeneous molecular landscape. In the pediatric context, the NUP98 gene is a frequent target of chromosomal rearrangements that are linked to poor prognosis and unfavorable treatment outcomes in different AML subtypes. The translocations fuse NUP98 to a diverse array of partner genes, resulting in fusion proteins with novel functions. NUP98 fusion oncoproteins induce aberrant biomolecular condensation, abnormal gene expression programs, and re-wired protein interactions which ultimately cause alterations in the cell cycle and changes in cellular structures, all of which contribute to leukemia development. The extent of these effects is steered by the functional domains of the fusion partners and the influence of concomitant somatic mutations. In this review, we discuss the complex characteristics of NUP98 fusion proteins and potential novel therapeutic approaches for NUP98 fusion-driven AML.</p

The MLL-Menin Interaction is a Therapeutic Vulnerability in NUP98 -rearranged AML

Chromosomal translocations involving the NUP98 locus are among the most prevalent rearrangements in pediatric acute myeloid leukemia (AML). AML with NUP98 fusions is characterized by high expression of HOXA and MEIS1 genes and is associated with poor clinical outcome. NUP98 fusion proteins are recruited to their target genes by the mixed lineage leukemia (MLL) complex, which involves a direct interaction between MLL and Menin. Here, we show that therapeutic targeting of the Menin-MLL interaction inhibits the propagation of NUP98-rearrranged AML both ex vivo and in vivo. Treatment of primary AML cells with the Menin inhibitor revumenib (SNDX-5613) impairs proliferation and clonogenicity ex vivo in long-term coculture and drives myeloid differentiation. These phenotypic effects are associated with global gene expression changes in primary AML samples that involve the downregulation of many critical NUP98 fusion protein-target genes, such as MEIS1 and CDK6. In addition, Menin inhibition reduces the expression of both wild-type FLT3 and mutated FLT3-ITD, and in combination with FLT3 inhibitor, suppresses patient-derived NUP98-r AML cells in a synergistic manner. Revumenib treatment blocks leukemic engraftment and prevents leukemia-associated death of immunodeficient mice transplanted with NUP98::NSD1 FLT3-ITD-positive patient-derived AML cells. These results demonstrate that NUP98-rearranged AMLs are highly susceptible to inhibition of the MLL-Menin interaction and suggest the inclusion of AML patients harboring NUP98 fusions into the clinical evaluation of Menin inhibitors.</p

The MLL-Menin Interaction is a Therapeutic Vulnerability in NUP98 -rearranged AML

Chromosomal translocations involving the NUP98 locus are among the most prevalent rearrangements in pediatric acute myeloid leukemia (AML). AML with NUP98 fusions is characterized by high expression of HOXA and MEIS1 genes and is associated with poor clinical outcome. NUP98 fusion proteins are recruited to their target genes by the mixed lineage leukemia (MLL) complex, which involves a direct interaction between MLL and Menin. Here, we show that therapeutic targeting of the Menin–MLL interaction inhibits the propagation of NUP98-rearrranged AML both ex vivo and in vivo. Treatment of primary AML cells with the Menin inhibitor revumenib (SNDX-5613) impairs proliferation and clonogenicity ex vivo in long-term coculture and drives myeloid differentiation. These phenotypic effects are associated with global gene expression changes in primary AML samples that involve the downregulation of many critical NUP98 fusion protein-target genes, such as MEIS1 and CDK6. In addition, Menin inhibition reduces the expression of both wild-type FLT3 and mutated FLT3-ITD, and in combination with FLT3 inhibitor, suppresses patient-derived NUP98-r AML cells in a synergistic manner. Revumenib treatment blocks leukemic engraftment and prevents leukemia-associated death of immunodeficient mice transplanted with NUP98::NSD1 FLT3-ITD-positive patient-derived AML cells. These results demonstrate that NUP98-rearranged AMLs are highly susceptible to inhibition of the MLL–Menin interaction and suggest the inclusion of AML patients harboring NUP98 fusions into the clinical evaluation of Menin inhibitors

SMARCA5 interacts with NUP98-NSD1 oncofusion protein and sustains hematopoietic cells transformation

BACKGROUND: Acute myeloid leukemia (AML) is characterized by accumulation of aberrantly differentiated hematopoietic myeloid progenitor cells. The karyotyping-silent NUP98-NSD1 fusion is a molecular hallmark of pediatric AML and is associated with the activating FLT3-ITD mutation in &gt; 70% of the cases. NUP98-NSD1 fusion protein promotes myeloid progenitor self-renewal in mice via unknown molecular mechanism requiring both the NUP98 and the NSD1 moieties.METHODS: We used affinity purification coupled to label-free mass spectrometry (AP-MS) to examine the effect of NUP98-NSD1 structural domain deletions on nuclear interactome binding. We determined their functional relevance in NUP98-NSD1 immortalized primary murine hematopoietic stem and progenitor cells (HSPC) by inducible knockdown, pharmacological targeting, methylcellulose assay, RT-qPCR analysis and/or proximity ligation assays (PLA). Fluorescence recovery after photobleaching and b-isoxazole assay were performed to examine the phase transition capacity of NUP98-NSD1 in vitro and in vivo.RESULTS: We show that NUP98-NSD1 core interactome binding is largely dependent on the NUP98 phenylalanine-glycine (FG) repeat domains which mediate formation of liquid-like phase-separated NUP98-NSD1 nuclear condensates. We identified condensate constituents including imitation switch (ISWI) family member SMARCA5 and BPTF (bromodomain PHD finger transcription factor), both members of the nucleosome remodeling factor complex (NURF). We validated the interaction with SMARCA5 in NUP98-NSD1+ patient cells and demonstrated its functional role in NUP98-NSD1/FLT3-ITD immortalized primary murine hematopoietic cells by genetic and pharmacological targeting. Notably, SMARCA5 inhibition did not affect NUP98-NSD1 condensates suggesting that functional activity rather than condensate formation per se is crucial to maintain the transformed phenotype.CONCLUSIONS: NUP98-NSD1 interacts and colocalizes on the genome with SMARCA5 which is an essential mediator of the NUP98-NSD1 transformation in hematopoietic cells. Formation of NUP98-NSD1 phase-separated nuclear condensates is not sufficient for the maintenance of transformed phenotype, which suggests that selective targeting of condensate constituents might represent a new therapeutic strategy for NUP98-NSD1 driven AML.</p

Dynamic clonal progression in xenografts of acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21

Intrachromosomal amplification of chromosome 21 is a heterogeneous chromosomal rearrangement occurring in 2% of childhood precursor B-cell acute lymphoblastic leukemia. There are no cell lines with iAMP21 and these abnormalities are too complex to faithfully engineer in animal models. As a resource for future functional and pre-clinical studies, we have created xenografts from intrachromosomal amplification of chromosome 21 leukemia patient blasts and characterised them by in-vivo and ex-vivo luminescent imaging, FLOW immunophenotyping, and histological and ultrastructural analysis of bone marrow and the central nervous system. Investigation of up to three generations of xenografts revealed phenotypic evolution, branching genomic architecture and, compared with other B-cell acute lymphoblastic leukemia genetic subtypes, greater clonal diversity of leukemia initiating cells. In support of intrachromosomal amplification of chromosome 21 as a primary genetic abnormality, it was always retained through generations of xenografts, although we also observed the first example of structural evolution of this rearrangement. Clonal segregation in xenografts revealed convergent evolution of different secondary genomic abnormalities implicating several known tumour suppressor genes and a region, containing the B-cell adaptor, PIK3AP1, and nuclear receptor co-repressor, LCOR, in the progression of B-ALL. Tracking of mutations in patients and derived xenografts provided evidence for co-operation between abnormalities activating the RAS pathway in B-ALL and for their aggressive clonal expansion in the xeno-environment. Bi-allelic loss of the CDKN2A/B locus was recurrently maintained or emergent in xenografts and also strongly selected as RNA sequencing demonstrated a complete absence of reads for genes associated with the deletions

Therapeutic targeting in pediatric acute myeloid leukemia with aberrant HOX/MEIS1 expression

Despite advances in the clinical management of childhood acute myeloid leukemia (AML) during the last decades, outcome remains fatal in approximately one third of patients. Primary chemoresistance, relapse and acute and long-term toxicities to conventional myelosuppressive therapies still constitute significant challenges and emphasize the unmet need for effective targeted therapies. Years of scientific efforts have translated into extensive insights on the heterogeneous spectrum of genetics and oncogenic signaling pathways of AML and identified a subset of patients characterized by upregulation of HOXA and HOXB homeobox genes and myeloid ecotropic virus insertion site 1 (MEIS1). Aberrant HOXA/MEIS1 expression is associated with genotypes such as rearrangements in Histone-lysine N-methyltransferase 2A (KMT2A-r), nucleoporin 98 (NUP98-r) and mutated nucleophosmin (NPM1c) that are found in approximately one third of children with AML. AML with upregulated HOXA/MEIS1 shares a number of molecular vulnerabilities amenable to recently developed molecules targeting the assembly of protein complexes or transcriptional regulators. The interaction between the nuclear scaffold protein menin and KMT2A has gained particular interest and constitutes a molecular dependency for maintenance of the HOXA/MEIS1 transcription program. Menin inhibitors disrupt the menin-KMT2A complex in preclinical models of KMT2A-r, NUP98-r and NPM1c acute leukemias and its occupancy at target genes leading to leukemic cell differentiation and apoptosis. Early-phase clinical trials are either ongoing or in development and preliminary data suggests tolerable toxicities and encouraging efficacy of menin inhibitors in adults with relapsed or refractory KMT2A-r and NPM1c AML. The Pediatric Acute Leukemia/European Pediatric Acute Leukemia (PedAL/EUPAL) project is focused to advance and coordinate informative clinical trials with new agents and constitute an ideal framework for testing of menin inhibitors in pediatric study populations. Menin inhibitors in combination with standard chemotherapy or other targeting agents may enhance anti-leukemic effects and constitute rational treatment strategies for select genotypes of childhood AML, and provide enhanced safety to avoid differentiation syndrome. In this review, we discuss the pathophysiological mechanisms in KMT2A-r, NUP98-r and NPM1c AML, emerging molecules targeting the HOXA/MEIS1 transcription program with menin inhibitors as the most prominent examples and future therapeutic implications of these agents in childhood AML.</p